Anti-asthmatic combinations comprising surface active phospholipids

ABSTRACT

Disclosed is a combination product for use in treating asthma and other respiratory conditions comprising a medicament comprising a surface active phospholipid composition in the form of a fine powder and an antiasthma drug. The product is arranged to be administered to the lungs by inhalation, for example, by the disclosed devices.

The present application is a nationalization of International PatentApplication PCT/GB99/03952, filed Nov. 26, 1999, which claims priorityto British Patent Application 9912639.3, filed May 8, 1999 and toInternational Patent Application PCT/GB98/03543, filed Nov. 26, 1998.

FIELD OF THE INVENTION

This invention relates to pharmaceutical products for use in thetreatment of asthma and to deliver devices including the products.

This invention relates to pharmaceutical products for use in thetreatment of asthma and to delivery devices including the products.

It has been estimated that asthma affects between 4 and 10 percent ofthe population, causing distress and alarm to both sufferers andbystanders. Asthma attacks appear to be precipitated in many cases by anumber of factors such as exercise or pollutants in the inspired air.Other agents such as pollen and airborne particles may predispose anasthma sufferer to an attack by sensitising the airways. This has led tothe belief that effective treatment should include administration ofdrugs which reduce the sensitivity of asthma sufferers to allergens orwhich neutralise the allergic reaction.

The lungs and airways of non-asthmatics may contain a natural protectivebarrier which prevents pollutants and other potential irritants fromreaching receptors which would otherwise produce an acute attack.Studies have suggested that it is possible to simulate in the lungs ofasthma sufferers the situation in normal lungs by causing surface-activephospholipids (SAPL) to bind to the tissue surface of the lungs, therebyreducing the number of receptors exposed to noxious stimuli and reducingthe broncho-constrictor reflex.

SAPLs are used clinically for the treatment of respiratory distresssyndrome (RDS) in neonates. In this role, it has been assumed that theSAPL functions by reducing the high surface tension forces at theair-water interface within the alveoli, thereby reducing the pressureneeded to expand the lungs, see Bangham et al., Colloids & Surfaces, 10(1984), 337 to 341. Thus, commercially available formulations of SAPLhave been designed co spread rapidly over an air-aqueous interface,thereby reducing what is otherwise a very high surface tension of water.

Limited clinical studies have been carried out to determine the effectof commercial SAPLs marketed for treatment of RDS in neonates onasthmatic subjects,—see Kurashima et al Jap. J. Allergol 1991; 40, 160.This paper reported some amelioration of bronchoconstriction inasthmatic adults using an SAPL obtained by extraction from bovine lungs.In another study on children, also using an SAPL obtained from bovinelungs, no significant changes in lung function or histamine responsewere found,—see Oetomo et al—American Journal of Respiratory andCritical Care Medicine 153; 1996, page 1148.

EP 0 528 034A describes the use of pulmonary surface active material asan ingredient of an antiasthmatic, which is in the form of a liquid orsuspension for injection or spraying into the patient's air way.

The invention provides a therapeutic combination product for use in theprevention and/or treatment of asthma comprising

(a) a medicament comprising a surface active phospholipid (SAPL)composition in finely divided form, the SAPL including a component whichenhances spreading of the medicament over a surface at about normalmammalian body temperature; and

b) an antiasthma drug;

wherein ingredients (a) and (b) are provided in a form foradministration together or separately.

It is believed that the finely divided powder of ingredient (a), whichpreferably comprises at least first and second components, has twoimportant effects:

First, the medicament (a) has surfactant properties, which enable it tospread rapidly over the surfaces of the lungs and air passages. It is animportant feature of the present invention that the medicament (a) is inthe form of a powder, that is, it is in solid form. The “dry” surfactanthas a high surface activity. It is believed that, on contact of a firstcomponent of the medicament (a) with the mucous within the lungs, thepresence of a second component results in a lowering of the meltingpoint of the first component, promoting rapid spreading of the firstcomponent over the liquid-air interface as a thin film at bodytemperature. For example, the normal melting temperature of dipalmitoylphosphatidyl choline, which is a preferred first component is about 40°C., that is, above the normal body temperature. When used in combinationwith a suitable second component, such as a phosphatidyl glycerol,however, the melting point of the dipalmitoyl phosphatidyl choline canin effect be reduced to below the normal body temperature.

Second, once the surface active medicament is in situ over the surfacesof the lungs and air passages, a component of the composition is thoughtto migrate across the mucous layer enabling a thin hydrophobic lining orcoating co be adsorbed onto the tissue surface. Thus, over and above thesurface tension reducing properties mentioned above the medicament ofthe invention is believed to provide a protective effect by virtue ofthe adsorbed layer in binding to the epithelium, the phospholipid maymask the irritant receptors which elicit the bronchorestrictor reflex,that is, which cause narrowing of the bronchi.

The medicament (a) is in finely divided solid form. It is believed that,as a consequence of the high surface activity of medicament (a) in thatform there results a significant drop in surface tension on contact withthe aqueous mucous layer of the lung, giving enhanced effectiveness ofingredient (a) and permitting improved access to the lung surfaces forthe antiasthma drug(s) to be administered. Thus, the use of themedicament (a) in combination with an antiasthma drug is believed toenhance the effectiveness of the antiasthma drug.

Moreover, as mentioned above, the binding of the phospholipid componentto the lung surface is believed to reduce bronchorestrictor as aconsequence of a reduction, in receptor-mediated activity attributableto the masking of irritant receptors. That reduced bronchorestrictionacts cumulatively with the anti-bronchorestrictive activity of theantiasthma drug. Thus, in some circumstances it may be possible fordosages of an antiasthma drug to be administered to a given patient tobe reduced, as a consequence of the synergistic effect of medicament (a)in enhancing the effectiveness of the antiasthma drug as well as theadditional anti-bronchorestrictive activity of medicament (a) itself.

“Finely divided” as used herein means that the material has a particlesize distribution which is such that at least a major proportion byweight of the particles are small enough to enter into a patient'sairways and, preferably, deep into the lungs when inhaled. In practice,the first and second components preferably each have a particle sizedistribution which is such that not less than 90%, by weight, of theparticles of those components in combination, and more preferably ofeach of the first and second components, have a particle size of notgreater than 10 μm, and especially of not greater than 5 μm.Advantageously, the median particle size of the combined first andsecond components, and more preferably of each of the first and secondcomponents is not more than 10 μm, and preferably not more than 5 μm.The median particle size may be less than 3 μm, for example, about 1.2μm. It may be desirable in some circumstances for the particles to havea median particle size of at least 0.5 μm. The size of the particles maybe calculated by laser diffraction, or by any other method by which theaerodynamic diameter of particles can be determined. “Median particlesize” as used herein means mass median aerodynamic diameter (“MMAD”).The MMAD may be determined using any suitable method, for example, usinga Multi-Stage Liquid Impinger in accordance with the method described inEuropean Pharmacopoeia (supplement 1999) 2.9.18 (Aerodynamic assessmentof fine particles). Alternatively, the size distribution of theparticles may be characterised by their volume mean diameter (VMD).Advantageously, the VMD is not more than 10 μm, for example not morethan 5 μm, and preferably less than 3 μm. Finely divided dry powders ofthis kind (which may be described as fumed powders) can be adsorbed ontothe surfaces of lung tissue and are believed, in use, to become bound tothe epithelium.

A finely divided solid mixture of said first and second components ofthe medicament (a) may be obtained by size reduction of larger particlesby any suitable size reduction method, preferably before mixing.Preferably, the first component of the medicament (a) comprises one ormore compounds selected from the group consisting of diacyl phosphatidylcholines. Examples of suitable diacyl phosphatidyl cholines (DAPCs), aredioleyl phosphatidyl choline (DOPC); distearyl phosphatidyl choline(DSPC) and dipalmitoyl phosphatidyl choline (DPPC). Each of thosecompounds appears to be capable of forming a thin film or coating onsurfaces of the lungs. Most preferably, the first component is DPPC.

The second component may comprise one or more compounds selected fromthe group consisting of phosphatidyl glycerols (PG); phosphatidylethanolamines (PE); phosphatidyl serines (PS); phosphatidyl inositols(PI) and chlorestyl palmitate (CP)

Phosphatidyl glycerol (PG) is believed to be capable of binding to lungtissue and possibly enhancing the binding of the first component and is,therefore, a preferred second component. PG is also a preferred secondcomponent because of its ability to form with the first component a veryfinely-divided, dry powder dispersion in air.

The medicament advantageously comprises a diacyl phosphatidyl cholineand a phosphatidyl glycerol. The phosphatidyl glycerol is advantageouslya diacyl phosphatidyl glycerol. The acyl groups of the phosphatidylglycerol, which may be the same or different, are advantageously eachfatty acid acyl groups which may have from 14 to 22 carbon atoms. Inpractice, the phosphatidyl glycerol component may be a mixture ofphosphatidyl glycerols containing different acyl groups. Thephosphatidyl glycerol is expediently obtained by synthesis from purifiedlecithin, and the composition of the acyl substituents is then dependenton the source of the lecithin used as the raw material. It is preferredfor at least a proportion of the fatty acid acyl groups of thephosphatidyl glycerol to be unsaturated fatty acid residues, forexample, mono- or di-unsaturated C18 or C20 fatty acid residues.Preferred acyl substituents in the phosphatidyl glycerol component arepalmitoleoyl, oleoyl, linoleoyl, linolenoyl and arachidonoyl. Themedicament preferably comprises dipalmitoyl phosphatidyl choline andphosphatidyl glycerol, with the phosphatidyl moiety of the phosphatidylglycerol advantageously being obtainable from the phosphatidyl moiety ofegg lecithin.

The first and second components of the medicament (a) may be present ina weight ratio of from 1:9 to 9:1. Advantageously, the proportion byweight of the first component exceeds that of the second component.Preferably, said first component and said second component are presentin a weight ratio of from 6:4 to 8:2. At a weight ratio of about 7:3,the mixture spreads rapidly at a temperature of 35° C. or above.

DPPC can be prepared synthetically by acylation ofglycerylphosphorylcholine using the method of Baer & Bachrea—Can. J. OfBiochem. Physiol 1959, 37, page 953 and is available commercially fromSigma (London) Ltd. The PG may be prepared from egg phosphatidylcholineby the methods of Comfurions et al, Biochem. Biophys Acta 1977, 488,pages 36 to 42; and Dawson, Biochem J. 1967, 102, pages 205 to 210. Whenco-precipitated with DPPC from a common solvent such as chloroform, PGforms with DPPC a fine powder which spreads rapidly over the surfaces ofthe airways and lungs. The most preferred composition of the inventioncontains DPPC and a phosphatidyl glycerol derived from egg phosphatidylcholine and having a mixture of C16, C18 (saturated and unsaturated) andC20 (unsaturated) acyl groups. One form of that composition isobtainable from Britannia Pharmaceuticals Ltd., 41-51 Brighton Road,Redhill, Surrey, under the trade mark “ALEC”. For use in the device ofthe present invention, however, it is preferred for the particle size ofthe mixture to be less than that of “ALEC” in the form in which it iscurrently obtainable commercially. To obtain a mixture in which theparticle size is suitable for use in the device of the invention, thephospholipid components may be dissolved in a suitable solvent, forexample ethanol, the solution filtered and vacuum-dried, and the solidproduct size-reduced to obtain particles of the desired size. Duringsize-reduction, care should be taken to protect the mixture frommoisture, oxygen, direct heat, electrostatic charge and microbialcontamination.

“Antiasthma drug” is used herein to include any drug which hasbiological activity against asthma. It will be appreciated that, as usedherein, “antiasthma drug” is to be understood as not including thecompositions of the medicament of ingredient (a). The antiasthma drugmay comprise one or more respiratory drugs including but not limited todrugs selected from the group consisting of β₂-agonists, steroids,cromones, antimuscarinic drugs and leukotriene receptor antagonists. Thecombination product may comprise one or more said antiasthma drugs in anamount of up to 10 parts, especially up to one part by weight perhundred parts by weight of said first and second components, incombination, of the said medicament (a). It will be appreciated that therespiratory drug or drugs should be present in such an amount that eachdose delivered by the device contains an effective amount of the drug ordrugs.

The combination product may comprise a β₂-agonist which may beterbutaline, a salt of terbutaline, for example terbutaline sulphate, ora combination thereof or may be salbutamol, a salt of salbutamol or acombination thereof. Salbutamol and its salts are widely used in thetreatment of respiratory disease. The active particles may be particlesof salbutamol sulchate. Long-acting β₂ adrenoceptor agonists may bepresent, for example, formoterol, salmeterol, and salts thereof.

The combination product may comprise an antimuscarinic drug, for exampleipatropium bromide.

The combination product may comprise a steroid, which may be, forexample, beclomethasone dipropionate, budesonide, triamcinoloneacetonide or may be fluticasone. The medicament may comprise otherprophylactic drugs, including cromones, for example, sodium cromoglycateor nedocromil. The medicament may include a leukotriene receptorantagonist.

Advantageously, at least ingredient (a) is arranged to be delivered to apatent in the form of at least one individual inhalable dose, the oreach individual dose comprising said first and second components ofingredient (a) in a combined amount of at least 10 mg. Whereasphospholipids have been disclosed previously as adjuvants in certainforms of delivery device, the amounts of phospholipid administered in adose by those previously disclosed devices have been much smaller thanthose envisaged according to the present invention. In fact, it ispreferred in accordance with the present invention for each individualdose to comprise at least 25 mg, and more especially at least 40 mg ofsaid first and second components. The first and second components aresubstantially non-toxic, and the upper limit of the dosage of ingredient(a) may therefore in general be selected having regard to conveniencetaking into account matters such as, for example, the comfort of thepatient and/or design parameters of the device. In general, however, thedevice will be suck that it can deliver doses of up to 1000 mg,advantageously up to 500 mg, preferably up to 200 mg, and especially upto 100 mg. Preferably, at least ingredient (a) is arranged forsequential delivery of a multiplicity of inhalable doses.

The products of the invention have the further advantage that the firstand second components of the medicament (a) may be of synthetic origin.It has been found undesirable to expose asthmatic patients to proteinsof animal origin, because such proteins can have a sensitising effect onsuch patients, and thus the use of synthetic material has considerableadvantages over the use of surfactants of animal origin that may containanimal protein.

Because it is desirable in the present invention to achieve a relativelylong term adsorption of the medicament (a) on the lung surface, it ishighly desirable that the medicament (or any active components) shouldnot break down in the environment of the lungs. One of the factors whichwill reduce the life of a lining or coating will be the presence ofenzymes, such as phospholipase A, capable of digesting DPPC and/or PG.Such enzymes only attack the laevorotatory (L) form, which constitutesthe naturally occurring form. Therefore, the medicament shouldpreferably contain the dextrorotatory (D) form or at least comprise aracemic mixture, which is obtained by synthetic routes. Suitabledispersion devices may employ a propellant such as a halocarbon to formthe gas stream and may include a tapered discharge nozzle baffle or aventuri to accelerate particles through a discharge nozzle, and toremove oversized particles. Suitable halocarbons includehydrofluorocarbons, hydrofluorochlorocarbons and fluorochlorocarbonshaving a low boiling point, such as those marketed under the trade mark“Freon”. The medicament may be packaged with a propellant in apressurised aerosol container within the inhaler. Other inhalers have animpeller which mixes the powder into an air stream and delivers thepowder-laden air into the patient's airways see, e.g. U.S. Pat. No.5,577,497.

A preferred method and apparatus for administering the medicament (a)involves dispersing the powdered medicament in a propellant gas stream.For example, a pressurised canister of a liquefied gas may be connectedto a vial containing the medicament. By releasing controlled amounts ofgas from the canister into the vial, increments of the medicament areejected from the vial as a cloud of powder and may be inhaled by theuser. Where compatible with the characteristics of the antiasthma drugto be co-administered, that drug may be introduced into the gas stream,so that it is administered in admixture with the medicament (a). It isenvisaged that, in use, one or two inhalable doses of the medicament(a), each dose containing 50 mg, may be administered up to three timesdaily.

Where the antiasthma drug is to be administered separately andsequentially with the medicament (a) administration of the antiasthmadrug may occur as and when required by the patient and the timing ofadministration may thus be independent of the timing of administrationof the medicament (a).

The present invention provides a delivery device for administering to apatient by inhalation a medicament for the prevention or treatment ofasthma, the delivery device containing a medicament comprising a surfaceactive phospholipid (SAPL) composition in finely divided form, the SAPLincluding a component which enhances the spreading of the medicament andthe delivery device being capable of delivering of at least oneindividual dose in an amount of at least 10 mg.

The invention also provides a delivery device for administering to apatient by inhalation a medicament for the prevention or treatment ofasthma, the delivery device containing a medicament, the medicamentbeing in finely divided powder form and comprising a first componentconsisting of one or more phosphacidyl cholines and a second componentconsisting of one or more compounds selected from the group consistingof phosphatidyl glycerols, phosphatidyl ethanolamines, phosphatidylserines, phosphatidyl inositols and chlorestyl palmitate, the deliverydevice being arranged for delivery of at least one individual inhalabledose, the or each individual dose comprising said first phospholipidcomponent and said second component in a combined amount of at least 10mg.

Furthermore, the invention provides use of (a) a surface activephospholipid (SAPL) composition in finely divided form conjointly with(b) an antiasthma drug in the manufacture of a medicament for thecontrol of asthma.

One form of dispenser according to the invention will now be describedin detail, by way of illustration, with reference to the accompanyingdrawings, in which:

FIG. 1 is a side elevation of a delivery device;

FIG. 2 is a similar view, but shows its interior; and

FIG. 3 is a schematic view of another embodiment of delivery device inaccordance with the invention.

In the drawings, a casing 1 is formed from two plastic mouldings 2 and 3which snap together to form a container for a pressurised canister 4 anda vial 5. Canister 4 contains a low boiling liquid, preferably ahydrofluorocarbon such as HFA-134a or HFC-227, under sufficient pressureto maintain the propellant liquid at normal room temperature. Vial 5contains the powdered medicament (a), such as “ALEC”. Canister 4 has arelease valve 6 which is received in a recess 7 so that finger pressureon the inverted end 8 of the canister will cause propellant to bereleased into a tube 9. Tube 9 is typically a hard plastics, e.g. pvc orpolypropylene, tube of about 2-3 mm outside diameter and about 0.5 to 2mm inside diameter. Tube 9 connects valve 6 with a fitting 10 and thenceto a tube or needle 11 which extends into the vial 5. Vial 5 may beclosed with a rubber seal which is penetrated by the tube or needle 11and self-seals around the tube or needle. A second needle or tube 12extends part way into the vial through the rubber seal in the neck ofthe vial and connects with a fitting 13. Fitting 13 discharges into amouthpiece 14 which is a comfortable shape for the user to place in themouth. When the patient is in need of medication, he places themouthpiece 14 into his mouth and breaths and simultaneously depressesthe canister 4. This causes a cloud of medicament to be dispensed intothe patient's airways. Fittings 10 and 13 may be valves. Valves 10 maybe set to permit measured quantities of propellant to enter the vial.Similarly, valve 13 may be set to release when the pressure in the vialreaches a predetermined level. It will be appreciated that the dispensercan be used one-handed in an analogous manner to a conventionalnebulizer.

The antiasthma drug may be administered separately from a separatedevice either immediately before or after administration of themedicament (a), or separately as required by the patient. The antiasthmadrug may be dispensed from any suitable form of inhaler device, such asa dry powder inhaler or pressurised metered dose inhaler. Such devicescontaining antiasthma drugs are well known and widely availablecommercially, and do not require further explanation.

Instead, in addition to the powdered phospholipid composition, the vial5 may incorporate other known pulmonary or respiratory medicaments suchas salbutamol, Beclomethasone, corticosteroids, or other asthma drugs.It is, however, preferred to package the conventional asthma drug in thepropellant canister or in a capsule interposed between the propellantcontainer and the vial containing the phospholipid composition. In thisway, the lungs and airways receive a cloud of phospholipid compositionand an aerosol of the conventional drug sequentially or simultaneously.This combined therapy gives both quick relief and lasting protection asthe film of phospholipid composition spreads over the lung tissue.Instead of packaging the phospholipid composition in a multi-use vial,it may be contained in a capsule, which may be a single use quantity,between the outlet from the propellant canister and the mouthpiece.

Another form of delivery device is illustrated in FIG. 3.

Conceptually, the device 101 shown in FIG. 3 provides a receptacle 102having a volume of several liters which is filled with aerosolubilizedsolid SAPL composition, optionally also including an antiasthma drug,and is then inhaled by a patient via a breathing tube 120 connected to apipe 104 leading from the receptacle. Receptacle 102 1s first evacuatedusing vacuum pump 115. A quantity of the solid, powdered SAPLcomposition is contained within a mesh type holder 105 within a tube106, and air is then introduced through the tube 106 to cause the SAPLpowder to form an aerosolubilized cloud within the receptacle 102. Whenreceptacle 102 reaches approximately atmospheric pressure, breathingtube 120 is opened to permit the patient to inhale the SAPL composition.

The device 101 comprises a stainless steel receptacle 102 of volumeapproximately 4 liters which has an aperture 103 at its top extremity towhich a vertically extending pipe 104 is connected. Pipe 104 isconnected to a transverse pipe 109 and also a breathing tube 120 whichextends through a screen 121, so that the apparatus is not visible tothe patient. Breathing tube 120 may be fitted with a plug at its distantend, the plug being removable before use. A mesh holder 105 is mountedon the top of the receptacle 102 as part of a connection between an airline 106 and the receptacle. The mesh holder can be disassembled tointroduce a quantity of powdered medicament into the delivery device.One end of the air line 106 is connected, via the mesh holder, to thereceptacle 102 via a port 103. The other end of the air line 106 isconnected, via control device 107, to a regulated source 108 ofcompressed is propellant, e.g. air. If desired, the source of compressedpropellant can also contain a biologically active component for thetreatment of asthma. The pipe 104 extends upwardly from receptacle tomeet a horizontally extending pipe 109, from one end of which thereextends pipe 110 to atmosphere. A valve 111, openable by means of ahandle 112, is provided in the horizontally extending pipe 109, closingoff the pipe 110 from the receptacle 102 except when valve 111 is open.

At the other end of the horizontal pipe 109 there is provided a pressuregauge 113. At that end, the horizontal pipe 109 is connected to an airline 114, which extends, via the control device 107, to a vacuum pump115, which is controllable independently of the control device 107. Avalve 116, operable by a handle 117, is provided for the purpose ofopening or closing the pipe between the receptacle 102 and the air line114.

A safety pressure relief valve 118 is incorporated in the apparatus andis preferably arranged to open at 0.034 bar above atmospheric pressure.

In use, micronised SAPL composition (optionally together with anantiasthma drug) may be introduced into the mesh holder device 105,which is then inserted into the port 103 leading into the receptacle102. On insertion of the mesh holder device, the receptacle is sealed,the valves 111 and 116 both being closed. The pressure inside thereceptacle 102 is then reduced by means of opening valve 116 and pumpingair out of the receptacle 102 through air line 114.

Control unit 107 may include a needle valve (which may be adjustable) cocontrol the rate at which air is evacuated from the receptacle 102. Ifpressure falls too rapidly in the receptacle, it may cause the powderedmedicament in the mesh holder device to be sucked prematurely into thereceptacle. Thereafter, the valve 116 is closed. Whilst the receptacle102 remains sealed at reduced internal pressure, the regulatedcompressed air source 108 is actuated temporarily to inject air into thereceptacle 102 through the mesh holder device 105. As a consequence, thepowder in the mesh holder device 105 becomes aerosolised and enters thereceptacle 102. The pressure may be monitored using the pressure gauge113 and should at this stage be at or slightly below atmosphericpressure.

The plug is then removed from the mouthpiece of the breathing tube andthe patient can then inhale the contents of the receptacle by sucking onthe mouthpiece end of the breathing tube.

After the inhalation step, the valve 111 may be closed, and the cyclerecommenced.

If desired, the quantity of the powder successfully aerosolised may bedetermined by weighing the mesh and powder before use (the weight of themesh previously having been determined) and weighing the mesh with anyresidual powder after use of the device.

As indicated above, an antiasthma drug may be present in the source ofcompressed propellant, or be placed in the mesh holder device with theSAPL.

If preferred, or if necessitated by the nature of the antiasthma drug tobe administered in a combination treatment with the surface activephospholipid composition, the antiasthma drug may be administeredseparately from another device, for example, a dry powder inhaler orpressurised metered dose inhaler of known kind widely available for headministration of antiasthma drugs.

Determination of Fine Particle Fraction of Phospholipid Composition

As already mentioned, finely divided ALEC for use in the products of theinvention may be obtained by dissolving, filtering and vacuum-drying thecomponents and size-reducing the solid product so obtained. The deliveryof the size-reduced material was monitored using a Multi-Stage Impinger(MLSI) in accordance with the method described in European Pharmacopoeia(supplement 1999), 2.9.18 (Aerodynamic assessment of fine particles).Vials of the material were loaded on the 5-stage MLSI and delivery ofthe material tested under a number of operating conditions. Each volumeof air drawn of 41 is considered equivalent to one patient inhalation.The results, in Table 1, showed that a relatively large respirablefraction was generated. The respirable (or fine particle) fractionrepresents particles which reach stages 3, 4 and 5 of the MSLI,indicating a particle size of less than about 5.3 μm. Such particles areconsidered to be of a size such that they would enter deep into the lungof a typical patient.

TABLE 1 Total Mass Mass of Delivered Mass of Flow Phos- into Phos- FineRate Volume pho- Device pho Fine Particle Through of Air lipid (mg)lipid Particle Fraction MSLI Drawn Loaded (% of mass MSLI Fraction (%ex- (L/min) (Litres) (mg) loaded) (mg) (mg) device) 100 4 L 179.1 150.3(84%) 55.1 46.5 84.5 100 2 × 4 L 169.2 132.5 (78%) 60.0 45.1 75.1 100 3× 4 L 162.0 133.1 (82%) 66.0 55.7 84.4 100 3 L 136.1 119.8 (88%) 58.645.1 76.9 100 4 L 129.7 107.9 (83%) 53.0 44.7 84.3 100 2 × 4 L 113.5 94.0 (83%) 46.1 37.6 81.7 100 4 × 4 L 387.6 340.4 (88%) 117.5 86.5 73.6

Determination of Surface Activity of Phospholipid Compositions

A 2 cm×2 cm platinized grey dipping plate is heated to cherry red usingthe flame from a Bunsen burner or similar torch. The plate is suspendedfrom an electronic balance capable of weighing up to 500 mg.

To calibrate the apparatus, a small reflon dish is filled with distilledwater at approximately 20° C. (room temperature) and placed on alaboratory jack just beneath the dipping plate. The dish is then raisedso that the dipping plate just breaks the surface of the water, evenlyalong the bottom edge. The meniscus drawn up the dipping place is usedto set the display of the pen recorder of the electronic balance to readabout 73 mNm⁻¹ (the air/water surface tension of water at 20° C.). TheTeflon dish is lowered, emptied, cleaned, dried and then filled withreagent grade methanol. The dipping plate is cleaned as described above.The dish is then raised so that the dipping plate just breaks thesurface of the methanol, evenly along the bottom edge. The meniscusdrawn up the dipping plate will cause the pen recorder to read about 22mNm⁻¹ (the air/methanol surface tension of methanol at 20° C.¹). TheTeflon dish is lowered and the dipping plate is cleaned as describedabove. A zero-reading should be obtained for the cleaned plate alone(i.e. suspended in air).

To obtain a quantitative measure of the surface activity of a material,the Teflon dish is warmed to about 37° C., filled with water at not morethan 37° C. and placed on a laboratory jack just beneath the cleanedplate. The dish is then raised so that the dipping plate just breaks thesurface of the water, evenly along the bottom edge. The meniscus drawnup the dipping plate will give a reading of about 70 mNm⁻¹ (theapproximate air/water surface tension of warm water). The material isapplied onto the surface of the water using a small spatula. The amountapplied should be sufficient to ensure that a complete monolayer hasbeen formed on the surface of the water, such that an excess (as smallfree-floating particles) can be observed. The surface tension shouldfall instantly, that fall being recorded by the pen recorder.Equilibrium surface tension readings are taken from the pen recorderafter about 1 minute. The temperature of the water in the Teflon dishshould be not less than 35° C. immediately after the reading is taken.

The term “high surface activity” as used herein with reference to anycomposition for use in accordance with the invention means that theequilibrium surface tension as measured in the above method, is at least10% lower than the surface tension before the composition is applied tothe water surface. In practice, the reduction in surface tensionobtainable using certain phospholipid compositions such as thosementioned above in illustration of medicament (a) may exceed 50%.

A component included in admixture with another material is to beunderstood as enhancing the spreading of the other material if, incarrying out the above method for determination of surface activityusing the mixture and, separately, using the other material alone, thetime taken for the equilibrium surface tension to be reached is shorterfor the mixture, as compared to the material alone.

The above method describes determination of surface activity at 37° C.It will be appreciated that, where reference is made herein to enhancingspreading at about normal mammalian body temperature, the method shouldbe carried out at about the normal body temperature of the relevantmammal, where that is not about 37° C.

The following Example illustrates the binding of a preferredphospholipid to the epithelium:

EXAMPLE Reagents

L-α-Phosphatidylcholine, 1,2—di[1-¹⁴C]palmitoyl in Toluene:Ethanol (1:1v/v), 114 mCi/mmol, 50 μCi in 2 mL (CFA604 B36, Amersham)

L-α-Phosphatidylcholine, dipalmitoyl (C16:0) (P-6267, Sigma)

DL-α-Phosphatidyl-DL-glycerol, dipalmitoyl (C16:0) (P-5650, Sigma)

Egg Phosphatidylglycerol (Batch 24756, Macfarlan Smith, Ltd.)

Sodium Chloride, 0.9%, B.P. (Baxter Healthcare)

Calcium Chloride (C-4901, Sigma)

Toluene (T-4428, Sigma)

Ethanol, AnalaR (10107.7Y, BDH)

NCS-II Tissue Solubilizer, 0.5N Solution (NNCS-502), Amersham)

OCS Organic Counting Scintillant (NOCS104, Amersham)

In preparation for the dispersions in which the epithelium would beincubated, stock solutions of the phospholipid components were preparedon the first day of Run 1. These solutions were as follows:

L-α-DPPC, 2.4 mg. mL⁻¹ in toluene:ethanol, 1:1

DL-α-DPPG, 3.0 mg. mL⁻¹ in toluene:ethanol, 1:1

Egg PG, 3.0 mg. mL⁻¹ in toluene:ethanol, 1.1

All of the above solutions were stored at 4° C. in glass vials, thethreads of which were sealed with teflon tape to minimise evaporation ofthe solvent. Each glass vial was then placed inside a second, tightlycapped glass vial. These solutions were used for each of the five runsin the trial. A solution of 200 mg. L⁻¹ CaCl² in 0.9% saline was alsoprepared on the first day of Run 2 and was used in each of Runs 2 to 5.

Equipment

Special Ultrasonic Cleaner, Model G112 SP1G (Laboratory Supplies Co.Inc., Hicksville, N.Y., U.S.A.)

VF2 Vortex (IKA-Labortechnik)

Shaking Water Bath, Model TSB2-201-A (Thermoline Scientific Equipment,Smithfield, Australia)

Contherm Series Five, Fan Forced Oven (Contherm Scientific Ltd. LowerHutt, N.Z.) TRI-CARB 2700TR Liquid Scintilation Analyser (PackardInstrument Co., Meriden, Conn., U.S.A.)

Ultrasonic Cleaner, Model FXPI2 (Unisonics Pty. Ltd. Sydney, Australia)

Bronchial Epithelium

To provide a source of bronchial epithelium, porcine lungs were obtainedfrom an abattoir within 24 h of death. The lungs had been scored at 4°C. since the time of death. The secondary bronchus was dissected fromthe right and/or left lungs. The exterior surface of the bronchus wastrimmed of all lung tissue, and the bronchus was further cut intosections having a known surface area of bronchial epithelium(approximately 0.5 cm×0.5 cm), leaving the epithelium and cartilageintact. The surface of the epithelium was rinsed with 0.9% saline toremove any mucus.

Where necessary sections of bronchial epithelium were stored in 0.9%saline at −20 for 3 to 7 days until required for use. The sections werethawed before use on the first day of each run.

For bronchial epithelium, a total of five runs were is completed. Eachrun consisted of three groups, as follows:

1. DPPC only

2. DPPC+DPPG

3. DPPC+eqgPG

Four dispersions were prepared on the first day of each run. All groupsreceived both 20.5 μL (3.3 μg) of ¹⁴C-L-α-DPPC and 5.5 μL (13.2 μg) ofunlabelled L-α-DPPC from the stock solutions. In addition, Group 2received 5.5 μL (16.5 μg) DL-α-DPPG, while the same quantity of egg PGwas added to Group 3. In Groups 2 and 3, the ratio of total DPPC to PGwas 1:1. The phospholipic component was mixed with 6.6 ml of 0.9% salinefor Groups 1, 2, and 3. All of the above listed volumes were used whenthere were two sections of epithelium in each treatment group. When thenumber of sections was increased, the volumes of all components wereincreased accordingly, keeping all quantities in the same proportions asabove. Table 2 summarises the additives to the incubation mixtures.

TABLE 2 Components of Incubation Dispersions Egg Group Saline¹⁴C-L-α-DPPC L-α-DPPC DL-α-DPPC PG 1 X X X 2 X X X X 3 X X X X

To solubilise the phospholipid components in the aqueous medium, each ofthe four incubation dispersions was sonicated for 45 min, then vortexedto mix for 1 min.

From each dispersion, two lots of 2.8 mL were transferred to two glassvials. A single section of epithelium was incubated in each of thesedispersions, so that there were four groups of two sections of bronchialepithelium in each group. Bronchial epithelium was taken from a singlepig on any given day of incubation. Incubation was at 37° C. for 24 h ina shaking water bath.

Aliquots of the Group 1 dispersion were transferred to glassscintillation vials and incubated at 37° C. in an oven for the 24 h.These aliquots were used as the standards for the calibration curve.Matching aliquots from the other group dispersions were also taken, andthe β-counts from these were compared with those from the group 1dispersion as a check that all dispersions contained the same quantityof DPPC.

On the second day of each run, the sections of epithelium were removedfrom the incubation dispersions and were each rinsed 20 times with 0.9%saline, warmed to 37° C. in a water bath, to remove any loosely adheringphospholipid. Care was taken not to mechanically disturb the mucosalsurface of epithelium. Each section of bronchial epithelium was thenremoved from the attached cartilage. The sections of epithelium werefurther cut into smaller pieces to aid the digestion of the tissue bythe solubilising agent which was added in a volume of 1.5 mL to theepithelium in scintillation vials. The same volume of solubiliser wasadded to each of the standard aliquots and to a blank. All vials weregently shaken to mix the contents and were warmed to 55° C. in afan-forced convection oven overnight (18-20 h).

On the third day of each run, 10 mL of organic counting scintillant wereadded to each scintillation vial, and these were vortexed to mix for 30s.

The β-counts of each sample and standard were measure a using a liquidscintillation analyser. A second count was conducted within 7 h of thefirst count. If the two counts were similar, only the first count wasused to construct the line of calibration and to quantify the samples.

From the line of calibration, the mass of ¹⁴C-DPPC adsorbed to eachsection of epithelium was calculated. To calculate the mass of totalDPPC adsorbed to each section, the mass of ¹⁴C-DPPC was multiplied by 5since the quantity of ¹⁴C-DPPC in each of the dispersions was ⅕ of thetotal amount of DPPC. The result is expressed in Table 2 as the totalamount of DPPC adsorbed per cm² of epithelium.

The results in Table 3 show that increased binding of DPPC to bronchialepithelium is observed in the presence of DPPG, but that the extent ofbinding is improved still further where Egg PG is used instead of DPPG.

While the present invention has been described with particular referenceto the treatment of human patients for asthma, it is possible that theinvention may also be applicable to the treatment of other pulmonarydiseases or conditions such as rhinnitis.

The combination product of the present invention may also be employed inthe treatment of pulmonary conditions in other mammals. An example isreactive airway disease in horses.

TABLE 3 Total DPPC Adsorbed to Bronchial Epithelium (μg/cm²) DPPCDPPC:DPPG, 1:1 DPPC:Egg PG, 1:1 0.341 0.501 0.878 0.299 0.321 0.7430.219 0.214 0.472 0.116 0.263 0.731 0.276 0.378 0.705 0.280 0.494 0.5290.528 0.355 0.836 0.192 0.419 0.792 0.340 0.294 0.986 0.321 0.362 0.791n 10 10 10 Mean 0.291 0.360 0.746 SD 0.110 0.093 0.153

What is claimed is:
 1. A therapeutic combination product for treatmentof asthma comprising: (a) a medicament comprising a surface activephospholipid (SAPL) composition in finely divided dry powder form, theSAPL composition including a component which enhances spreading of themedicament over a surface at about normal mammalian body temperature andcomprising a first component comprising one or more phosphatidylcholines and a second component comprising one or more compoundsselected from the group consisting of phosphatidyl gylcerols,phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl inositolsand cholesterol palmitate, and (b) an antiasthma drug, whereiningredients (a) and (b) are provided in a form for administrationtogether and at least ingredient (a) is arranged for delivery of atleast one individual inhalable dose, the individual dose or eachindividual dose comprising said ingredient (a) in an inhalable amount ofat least 25 mg, for forming an adsorbed layer of phospholipid extendingover the surfaces of the lungs and air passages.
 2. A combinationproduct as claimed in claim 1, in which medicament (a) comprises saidfirst component and said second component in a weight ratio of from 1:9to 9:1.
 3. A combination product as claimed in claim 2, in which theproportion by weight of said first component exceeds that of said secondcomponent.
 4. A combination product as claimed in claim 3, in which saidfirst component and said second component are present in a weight ratioof from 6:4 to 8:2.
 5. A combination product as claimed in claim 1, inwhich the medicament (a) comprises a phosphatidyl glycerol.
 6. Acombination product as claimed in claim 5, in which the phosphatidylglycerol comprises one or more diacyl phosphatidyl glycerols, of whichat least a proportion of the acyl groups are unsaturated.
 7. Acombination product as claimed in claim 1, in which the medicament (a)comprises one or more compounds selected from the group consisting ofdiacyl phosphatidyl cholines.
 8. A combination product as claimed inclaim 7, in which the medicament (a) comprises dipalmitoyl phosphatidylcholine.
 9. A combination product as claimed in claim 1, in which themedicament (a) is in micronised form.
 10. A combination product asclaimed in claim 1, in which said medicament (a) has a median particlesize not exceeding 10 μm.
 11. A combination product as claimed in claim10, in which said medicament (a) has a median particle size notexceeding 5 μm.
 12. A combination product as claimed in claim 11, inwhich said medicament (a) has a median particle size of less than 3 μm.13. A combination product as claimed in claim 1, in which the antiasthmadrug comprises one or more respiratory drugs selected from the groupconsisting of β₂-agonists, steroids, cromones, antimuscarinic drugs andleukotriene receptor antagonists.
 14. A combination product as claimedin claim 13, which comprises one or more of said antiasthma drugs in anamount of up to 10 parts by weight per hundred parts by weight of saidfirst and second components of medicament (a) in combination.
 15. Acombination product as claimed in claim 14, which comprises one or moreof said respiratory drugs in an amount of up to one part by weight perhundred parts by weight of said first and second components ofmedicament (a) in combination.
 16. A combination product as claimed inclaim 13, in which ingredient (b) comprises a β₂-agonist.
 17. Acombination product as claimed in claim 13, in which ingredient (b)comprises a steroid.
 18. A combination product as claimed in claim 13,in which ingredient (b) comprises a cromone.
 19. A combination productas claimed in claim 13, in which ingredient (b) comprises a leukotrienereceptor antagonist.
 20. A combination product as claimed in claim 13,in which ingredient (b) comprises an antimuscarinic drug.
 21. Acombination product as claimed in claim 1, wherein the individual doseor each individual dose comprises said ingredient (a) in an inhalableamount of at least 40 mg.
 22. A combination product as claimed in claim1, in which the antiasthma drug is arranged for delivery in admixturewith ingredient (a).
 23. A method of treatment of asthma, comprisingadministering to a patient at least one dose of a combination product asdefined in claim 1.